Embryonic stem (ES) cells implicitly promise wonderful things for human health such as the replacement of damaged tissue. This need is great in the central nervous system (CNS) where regeneration is minimal. However, there is much to be learned before neural systems can be replaced. In addition to the goal of restoration, ES cells can provide in vitro model systems of neural differentiation and function in a way that has never before been achieved due to the complexity of neural tissue. The serotonin (5HT) neural system plays a pivotal role in mood, affective regulation in response to stress, integrative cognition, aggression, satiety and numerous other autonomic functions. In addition, serotonin per se is a critical factor in the developmental organization of the central nervous system and serotonin is present at a very early stage of fetal development. Gestation in primate species is quite different from that of rodents and a primate model is needed to investigate developmental questions about serotonin neurons. However, the use of pregnant macaques is expensive, time consuming and labor intensive. Thus, a cell culture model system would be of enormous import, but no primate or human immortalized serotonin cell line is available. We have utilized rhesus monkey embryonic stem cells (ESC) and induced differentiation into cultures that are 95% serotonin neurons with characteristics of mature rhesus serotonin neurons. We propose to determine similarities and differences between ESC-derived serotonin neurons and serotonin neurons in the macaque CNS, to use these cultures to examine basic questions of function and to determine the effects of placenta! steroid hormones on ESC-derived serotonin neural differentiation and function. Aim 1 will establish that rhesus ESC-derived serotonin neurons closely resemble primate CNS serotonin neurons; Aim 2 will compare the expression profile of ESC-derived serotonin neurons and Laser Captured CNS serotonin neurons for examination of similarities and differences in genes and pathways. A multidisciplinary approach including immunocytochemistry, qRT-PCR, in situ hybridization, serotonin uptake and release, electrophysiology, and gene expression profiling will be used to characterize, optimize and manipulate the differentiation of rhesus ES cells into serotonin neurons prior to grafting. The knowledge obtained from this work will lead to novel treatments for psychoneuropathologies. [unreadable] [unreadable]